ABSTRACT Traumatic brain injury (TBI) is an expanding public health epidemic with complex pathophysiology that is difficult to diagnose and thus treat. TBI blood testing should provide simple, reliable, real-time, objective diagnostic monitoring and outcome prediction across the entire severity spectrum. Neurotrauma biomarkers with insight into underlying TBI processes can address these needs. At UCLA, Dr. Wanner?s team has discovered and confirmed new Astrocyte Injury-Defined (AID) biomarkers for improved TBI patient assessment. Aldolase C (ALDOC) and brain lipid binding protein (BLBP) are elevated hyper-acutely and over prolonged periods after TBI. They are also robustly present after mild TBI. These two markers are linked to trauma-specific cell membrane wounding (mechanoporation) and glial fiber damage. In contrast, new small breakdown products (BDPs) of GFAP are selective for cell death and associate with lesions and poor outcome. The work?s long-term objective is to improve TBI assessment by profiling biofluid signatures for specific TBI manifestations using biomarkers defined by cellular trauma processes. This project proposes the development and validation of diagnostically useful, noninvasive AID biomarker assays for improved clinical stratification of TBI patients. The first aim is to design and optimize enzyme-linked immunosorbent and electrochemi- luminescence-based platform assays. An already created ALDOC assay prototype will be fully authenticated, while assays for BLBP and small GFAP-BDPs are being developed, using best-performing antibody pairs. AID markers have high brain selectivity that will be validated in tissue panels. Assay targets will be defined, and affinity for biomarker BDPs determined. Assay accuracy will be optimized for TBI discrimination and distinction from healthy and non-TBI trauma controls. The second aim will clinically validate AID biomarkers and determine their longitudinal biofluid kinetics. Transition between cerebrospinal fluid and circulation will be monitored and degradation determined after severe TBI. Diagnostic performance of AID markers will then be assessed in a large mild TBI cohort and correlated with lesion presence and symptomatic recovery. Mild TBI AID marker signals will be differentiated from healthy and orthopedic trauma controls. Critical concussion diagnosis is addressed by measuring AID markers in two well-defined cohorts of concussed football players alongside healthy controls, non-concussed and chronically impact-exposed players. AID marker blood levels will be correlated with MR imaging for diffuse white matter damage, blood-brain barrier permeability and blood flow changes. Different trauma kinetics and sensitivities of ALDOC, BLBP, and GFAP with its BDPs complement each other in multivariate analyses, offering superior assessment. Thus, AID markers will improve diagnostic performance because they reflect acute traumatic tissue wounding and compromise in addition to tissue loss. This work will benefit neurotrauma research and translation, as these novel tests have great potential to improve patient stratification in clinical trials and diagnostic monitoring of mild TBI patients.